Settling moths are the vital component of pollination in Himalayan ecosystem of North-East India, pollen transfer network approach revealed

Majority of the pollination related studies are based on the diurnal pollinators, and the nocturnal pollinators received less scientific attention. We reveal the significance of settling moths in pollination of angiosperm families in Himalayan ecosystem of North-East India. The refined and novel method of pollen extraction from the proboscides provides a more robust assessment of the pollen carrying capacity. The study is based on one of the largest data sets (140 pollen transporter moth species (PTMS)), with interpretation based on seasonal as well as altitudinal data. In the present study about 65% moths (91 species) carried sufficient quantities of pollen grains to be considered as potential pollinators (PPMS). Teliphasa sp. (Crambidae) and Cuculia sp. (Noctuidae) are found to carry the highest quantity of pollen. We found pollen grains of 21 plant families and the abundant pollen are from Betulaceae, Fabaceae, Rosaceae and Ericaceae. Species composition of PTMS and PPMS in pre-monsoon, monsoon, and post-monsoon revealed the dominance of Geometridae. Maximum diversity of PTMS and PPMS is found from 2000 to 2500 m altitude. The nocturnal pollen transfer network matrices exhibited high degree of selectivity (H2ʹ = 0.86).

Pollination is an indispensable ecological process for the continuity of germplasm 1 and is mainly driven by wind, water and animals as vectors 1,2 . About 87.5% of all angiosperms are mainly pollinated by animals [3][4][5][6][7] . Among these, pollinating insects have important mutualistic relationships with angiosperms, which are essential for the conservation of wild as well as agricultural landscapes 2,8 .
So far, most research on pollinating insects has focused on diurnal pollinators, and the nocturnal pollinators have traditionally received less scientific attention 9 . Despite recent research in the last many years on their role in pollination, many questions left unanswered. An important review by Macgregor and co-workers revealed that 289 species of plants (mostly angiosperms from the orders Caryophyllales, Ericales, Gentianales, and Lamiales) under 75 families are partially or entirely pollinated by 21 moth families 8 . Some limited studies related to the comparable importance of the moths as pollinators to that of diurnal pollinators [10][11][12] , advocate the benefits of moths over the diurnal pollinators through mechanisms such as (1) longer-distance dispersal of pollen 13,14 , (2) good seed set despite deposition of fewer pollinia [15][16][17] and (3) increased pollination efficiency due to pollen deposition in a single visit 18 .
Among the moths, the settling moths (mainly of families other than Sphingidae, which hover when feeding from flowers) are extremely common and diverse flower visitors 19 but are less studied for their role in pollination. Many studies have focused on pollination by Sphingidae (i.e. the Sphingophily) [20][21][22][23][24][25][26][27][28][29][30][31] . The role of settling moths as pollinators i.e. the Phalaenophily 29 has also been studied, but arguably to a lesser degree 19,27 . Settling moths may potentially be effective pollinators of generalist plants which are not visited by hawkmoths or where the hawkmoths are not common 32 .
The noteworthy studies related to pollination by settling moths are sporadic, few and limited to certain regions of the world viz. in eastern England 12 , East Asia 33,34 , North Europe and America 35,36 . Other studies conducted on the pollination of plant families, Asteraceae, Ericaceae, Plantaginaceae, Myrtaceae, Campanulaceae, Thymelaeaceae, revealed the participation of settling moths 12,27,37,38 .
The Indian moths are negligibly studied for their role in pollination and only four papers could be reviewed for this aspect. Paul 39 studied the pollination efficiency of noctuid moth species in urban areas of Delhi. Sarkar and Sreedevi 40 studied the nocturnal insect pollinators of bottle gourd and ridge gourd crop of Andhra Pradesh and reported three species of settling moths, Arthroschista hilaralis (Crambidae), Diaphania indica (Crambidae) and Anadevidias peponis (Noctuidae), as major pollinators. The seasonal dynamics of plant-pollinator networks in agricultural landscapes of West Bengal and the pollination of medicinal plants in Tripura revealed the participation of settling moths 41,42 . The aim of the present study is to investigate the role of settling moths in pollination of angiosperm families distributed in Himalayan ecosystem of North-East India by quantifying various network level indices along with the effect of various seasons and altitudinal gradient on the pollen carrying capacity. The pollen transport does measure pollination success, but is a proxy whereby we can begin to assess involvement in pollination process 43 . The present study is based on the dataset generated from the light trapping at 24 field sites in Himalayan ecosystem of North-East India, over the period of 13 months.

Discussion
Our results are based on one of the largest data sets (140 species) interpreting the role of settling moths in pollination and is the first of its kind to understand the settling moths-plant pollination interactions in the central and eastern Himalayan ecosystem of India. Our approach of using the proboscis by isolating from the moth head and relaxing it for pollen extraction, provides a more robust assessment of the pollen carrying capacity of the moths, because (1) by using the proboscis for the pollen collection, the likelihood of contamination due to pollen rain is reduced (as swabbing of other parts of body result in collection of extra pollen grains from the body, unrelated to flower visitation), and (2) de-coiling the proboscis enables the study of pollen grains stuck within the coiling of the proboscis (which may remain there during swabbing). We found that substantial proportion (about 65%) of PTMS carried sufficient quantities of pollen grains (five or more than five) to be considered as potential pollinators (PPMS). Geometridae and Erebidae turned out to be the most important for the pollen transportation in the Himalayan region and on the other hand, pollen grains of Betulaceae, predominantly a wind-pollinated plant family, are transported in maximum. In our analysis, we include the Betulaceae related data as some wind-pollinated plant families may benefit from enhanced dispersal by insects, although the extent of this relationship is unknown 45 . Some recent studies reveal that some of the insect pollinators, particularly bees and syrphid fly species, collect pollen from a broad range of windpollinated plant species [46][47][48] , and also, some other studies [49][50][51][52][53][54][55][56][57] identified insect pollination in plant species which were presumed to be wind-pollinated. Our study may open some new horizons in the field of plant-pollinator interactions, a field with large gaps in knowledge. The present study reveals that the season and altitude affect the role of moths as PTMS greatly. In sub-tropical zone (1000-2000 m), we found highest number of PTMS as well as the diversity of pollen grains in post-monsoon whereas in temperate zone, the maximum diversity of PTMS and pollen grain is reported in pre-monsoon. The number of PTMS, PPMS and the diversity of pollen grains are lowest in the monsoon season. This may be due to the heavy rainfall with tropical rainstorms restricting the moth activities, as the weather parametres like rainfall, daily temperature plays a significant role in moth abundance, richness in a particular habitat 58 . Overall, we found maximum diversity of PTMS from higher sub-tropical zone (1500-2000 m) (44 PTMS) to lower temperate zone (2000-2500 m) (65 PTMS), a Himalayan zone with high floral diversity [59][60][61] . The tropical zone of Himalaya is mainly dominated with plant families like Fabaceae, Betulaceae, Euphorbiaceae, Theaceae and Asteraceae 61 , among which most of the species are trees. Whereas, in sub-tropical and temperate zone the floral component is characterized by plant families like Elaeocarpaceae, Oleaceae, Solanaceae, Rubiaceae, Zingiberaceae, Verbenaceae, Ericaceae, Asteraceae, Rosaceae and Orchidaceae 59,60,62,63 with dominance of shrubs and herbs. In our study, higher proportion of pollen from four plant families i.e., Fabaceae, Betulaceae, Ericaceae and Rosaceae is a possible indication of high preferences of settling moths towards these plant families, mainly for nectaring or may be for some non-nectar resource. Some non-lepidopteran pollinators visit wind-pollinated plants to collect or feed on pollen, and for non-floral resources i.e. insect honey dew, plant resins or resinous secretions 45,64-67 but our understanding about the attraction of settling moths towards small flowers with a very low quantity of nectar is very limited and requires further exploration.
We constructed the pollen number based network following Banza et al. 68 (with selectivity value of 0.79), and our network matrices also revealed high degree of selectivity (0.86) of settling moths in Himalayan ecosystem, which is highest value as compared to the other studies conducted on nocturnal moths in several ecosystems around the world 12,68-70 , thus, showing the importance of our study conducted in a larger spatial scale. The PDI score revealed that major proportion (98 species; 70%) of PTMS are specialist and only 42 species (30%) are generalists. High degree of specialism in moths is a strong indication of inter-dependency, and there are high possibilities that the dwindling moth diversity will adversely affect the plant diversity, and the other way round also. In our consolidated network, the value of interaction strength asymmetry (0.50) indicates that the specialised species are interacting with the generalised species and the vice versa.
We found significant correlation between the polyphagous nature of Achaea janata (Erebidae: Erebinae) and its ability to transfer pollen of different plant families. A. janata, commonly known as Castor oil semi-looper or Croton caterpillar, is a widely distributed species in the region and more broadly, it is a well-known pest of various economically important plants like Arachis hypogaea (Fabaceae), Rosa chinensis (Rosaceae), Tamarindus indica (Fabaceae), Glycine max (Fabaceae), Vigna mungo (Fabaceae), and Dalbergia sisso (Fabaceae) 71 . However, our results establish A. janata as a potential pollinator of three plant families, an addition to the knowledge provided by few such studies 72 indicating that the moths can provide net benefits as pollinators even when acting as larval herbivores of the same species. The phenomenon revealed that the species-interactions are much more complicated than we think of.
We believe that our results will lay a strong foundation for the studies related to moth-plant interaction, particularly in the Indian context and will further strengthen the concepts of ecosystem conservation rather than concentrating on few taxa. The present dataset is a tip of an iceberg as there are about 12,000 moth species in India 73 and about 160,000 in the world 74 and their abundance is still unknown to the science. We are quite sure that data generation and accumulation of knowledge through such type of studies will definitely strengthen our understanding about the role of nocturnal insect pollinators and will help in a better way regarding the management decisions for conservation, biodiversity, and agriculture.

Methods
Moth collection and sampling design. Representatives of settling moth families were collected from September, 2018 to October, 2019 in three different seasons i.e., pre-monsoon (May-June), monsoon (July-September) and post-monsoon season (October-November), using vertical sheet light traps operating with 160-W mercury vapour lamps from 24 randomly chosen sampling sites of Arunachal Pradesh, North Bengal and Sikkim ( Fig. 7; see The three clearly distinct seasons of Himalayan region were sampled as they greatly affect the floral and faunal components of the area under study 76 . The collections were made from a very large altitudinal gradient covering up to 3000 m and due to which, we interpreted our data in consolidate, as well as seasonally (pre-monsoon, monsoon and post-monsoon) and altitudinally (tropical zone (0-1000 m), sub-tropical zone (1000-2000 m) and temperate zone (2000-3000 m)).

Isolation of moth proboscis and light microscopy of Fuchsin jelly slides.
We used proboscides (c.a. 1800) to investigate the pollen loads (not the other parts of the body like head or legs) and also, for the first time, we separated the proboscis of each moth and directly processed it for the study of pollen (rather than swabbing). Our method reduced the chances of contamination due to pollen rain and it also enabled the study of those particular pollen grains which are found within the coiling of the proboscis, which may remain unstudied during swabbing. We isolated the proboscis from each moth using sterilised forceps. Each proboscis was kept in a separate vial with code number. To relax, the proboscis of each moth was placed on a glass slide and treated with few drops of 1:1 ratio of phenol and glycerol solution for 1-2 min. Afterwards, one to two drops of 50% solution of basic Fuchsin dye were added and the slide was mounted with cover slips and sealed with nail varnish. A Nikon 50i microscope with DP-25 digital camera (40 X magnification) was used to photograph the slides and to count the pollen grains. SEM analysis of isolated proboscis and pollen. The isolated proboscides of some specimens were also scanned with SEM, and photographs of pollen were taken using ZEISS Evo18 image acquiring software v5.09.
Moth and pollen grain identification. Moths were identified using various available literature, publications [77][78][79][80][81][82] and websites viz. www. moths ofind ia. org 83 , www. inatu ralist. org 84 . The classification followed here is given by Nieukerken 85 . Pollen grains were identified from palynological literature, books and websites including: www. paldat. org 86 , www. globa lpoll enpro ject. org 87-91 . Pollen transfer network construction and calculation of network matrices. Seven matrices (one consolidated, and two separate matrices (for sub-tropical and temperate zone) for each season) (see supplementary file 2) are prepared by keeping all the settling moth species along the rows and the plant families along the columns. The number of pollen grains from each plant family found on individual moth species is taken as the value for constructing the mutualistic network 44 . For quantification of all the mutualistic network parametres 'bipartite' package of RStudio is used 70 . The network-level metrics are (1) connectance (reflects the possible recorded links in the network or standardised number of species combinations) 92 , higher value indicates more connectance, (2) nestedness (range 0-100, higher value reflects more complexity of the network) 93 , (3) specialization or H 2 ' (range 0-1, higher value indicates more specialist species are interacting against the generalist species) 93,94 , (4) links per species (quantify links of species present in a mutualistic network), (5) interaction evenness (evenness of interactions of animals in a network based on Shannon's diversity, higher value reflects more evenness) 95 , (6) Shannon's diversity (to calculate the interaction richness of the network, high value indicates www.nature.com/scientificreports/ higher diversity), (7) interaction strength asymmetry (quantify weather the specialised species are interacting with generalised species in other level or vice versa) 96 , (8) linkage density (measures the density of linkage in a network) 97 and (9) species-level paired difference index (PDI) (measured to show the specialization of individual species for commonly visited plant family) 98 . The PDI score ranges from 0 to 1, 0 indicate complete generalism and 1 indicate total specialism in the plants' relationship with moths. www.nature.com/scientificreports/